Biochem Genet
DOI 10.1007/s10528-012-9564-7
Molecular Characterization of Primary Gene Pool
of Chickpea Based on ISSR Markers
Pooja Choudhary • Suruchi M. Khanna •
Pradeep K. Jain • Chellapilla Bharadwaj
Jitendra Kumar • Pramesh C. Lakhera •
Ramamurthy Srinivasan
•
Received: 13 January 2012 / Accepted: 30 August 2012
Ó Springer Science+Business Media New York 2013
Abstract Genetic diversity and relationships within and among members of the
primary gene pool of chickpea, including 38 accessions of Cicer arietinum, six of
C. reticulatum,, and four of C. echinospermum, were investigated using 31 ISSR
markers. The study revealed moderate diversity, detecting 141 fragments, of which
79 (56%) were polymorphic. Averages were 0.125 for polymorphic information
content, 0.350 for marker index, and 0.715 for resolving power. The UPGMA
dendrogram and the principal coordinate analysis revealed a clear differentiation
between wild and cultivated accessions. The clustering pattern did not strictly
follow the grouping of accessions by geographic origin but was in good agreement
with the pedigree data and the seed type. The study demonstrates that ISSRs provide
P. Choudhary S. M. Khanna P. K. Jain R. Srinivasan (&)
National Research Center on Plant Biotechnology, Pusa Campus, New Delhi 110012, India
e-mail: [email protected]
P. Choudhary
e-mail: [email protected]
S. M. Khanna
e-mail: [email protected]
P. K. Jain
e-mail: [email protected]
P. Choudhary P. C. Lakhera
Department of Biotechnology, Hemwati Nandan Bahuguna Garhwal University, Srinagar, Pauri
Garhwal 246 174, Uttarakhand, India
e-mail: [email protected]
C. Bharadwaj J. Kumar
Division of Genetics, Indian Agricultural Research Institute, Pusa Campus, New Delhi 110012,
India
e-mail: [email protected]
J. Kumar
e-mail: [email protected]
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Biochem Genet
promising marker tools in revealing genetic diversity and relationships in chickpea
and can contribute to efficient identification, conservation, and utilization of
germplasm for plant improvement through conventional as well as molecular
breeding approaches.
Keywords Chickpea Genetic diversity Molecular markers Principal coordinate analysis
Introduction
Chickpea (Cicer arietinum L.), a self-pollinated, diploid (2n = 29 = 16), coolseason pulse crop with a genome size of 740 Mbp, is widely grown in more than 50
countries representing all the continents (Upadhyaya et al. 2011). In addition to
being an excellent source of nutritive dietary protein for undernourished people
throughout the third world, chickpea plays an important role in improving soil
health, fertility, and sustainability of agro-ecosystems. Worldwide, it is the third
most important legume crop in terms of gross production (10.94 Mt) and acreage
(11.99 Mha), after soybeans (261.58 Mt, 102.39 Mha) and dry beans (23.23 Mt,
29.98 Mha). Over 95% of the area, production, and consumption of chickpea is in
developing countries, and the majority of the world’s chickpea crop is grown in
South Asia and the Mediterranean region. India is the largest producer, with an
estimated annual production of 7.48 Mt from an area of 8.2 Mha (FAO 2010).
The average global chickpea yield, 0.9 t/ha (FAO 2010), is far below its
presumed potential of 5 t/ha (Sudupak et al. 2002), and efforts to improve the
productivity of this crop by conventional breeding means have not been very
effective. Several biotic and abiotic stresses, its narrow genetic base, and a lack of
adapted varieties contribute to limited progress in the improvement of chickpea
yield (Millan et al. 2006). Improving resistance to biotic stresses and tolerance of
abiotic stresses, coupled with superior yield, are major aims of chickpea breeders.
Cultivated chickpea germplasm lacks the diversity that may include traits needed
for effective improvement of the crop. A number of wild annual species, especially
Cicer reticulatum and C. echinospermum, have drawn the attention of breeders,
since they harbor many agronomically desirable traits and are cross-compatible with
C. arietinum. As an important grain legume, investigation and management of the
genetic diversity and relationships within and between the cultivated chickpea and
its wild relatives are an obvious necessity to identify new sources of germplasm
bearing valuable genes.
Traditionally, a number of marker systems such as plant morphology, crossability
data, karyotypes, seed storage protein analysis, and enzymes have been used to
study the relationships among the Cicer species (Croser et al. 2003). Subsequently,
DNA-based markers such as RFLP (Udupa et al. 1993), RAPD (Sudupak et al.
2002; Ahmad et al. 2010), AFLP (Nguyen et al. 2004; Talebi et al. 2008), SSR
(Choumane et al. 2000; Upadhyaya et al. 2008; Sefera et al. 2011; Choudhary et al.
2012), and ISSR (Sudupak 2004; Rao et al. 2007; Bhagyawant and Srivastava 2008)
were developed and used to study genetic diversity and species relationships in
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Biochem Genet
chickpea. Most of the studies based on RFLP, RAPD, and AFLP reported abundant
diversity in wild but narrow genetic variation in cultivated chickpea. Studies based
on microsatellites or SSRs detected higher levels of polymorphism in cultivated
chickpea (Upadhyaya et al. 2008; Sefera et al. 2011; Choudhary et al. 2012).
Detection of variation at SSR loci, however, is technically demanding and relatively
expensive. An alternate approach is inter-simple sequence repeat (ISSR) based on
amplification of genomic DNA segments flanked by the inversely oriented SSR loci
at an amplifiable distance, which circumvents these disadvantages (Rafalski et al.
1996; dos Santos et al. 2011). The technique involves the use of a microsatellite
core unit bearing oligonucleotide primers, usually 16-25 bp long, nonanchored or
anchored at the 50 or 30 end with 1-4 degenerate nucleotides; it is fast and costefficient and does not require prior sequence knowledge. ISSRs are inherited in
simple Mendelian fashion, they segregate mostly as dominant markers (Ratnaparkhe
et al. 1998), and unlike RAPDs, they are highly reproducible and polymorphic
because they use relatively longer semiarbitrary SSR primers at high stringency
PCR conditions (Rafalski et al. 1996; Reddy et al. 2002).
Efforts to investigate genetic diversity and relationships in chickpea germplasm
using ISSR markers are skimpy (Rao et al. 2007; Bhagyawant and Srivastava 2008),
limited either by the small number of accessions used or the loci analyzed. The
present study was thus undertaken to analyze genetic diversity and relationships
within and between the popular chickpea cultivars and breeding lines and two of its
closest wild relatives (primary gene pool) using ISSR markers. The study provides
information about evolutionary relationships or the gene flow between the cultivated
chickpea and its wild relatives and will therefore serve as a useful indicator to
breeders and molecular biologists to select and use diverse accessions for varied
applications in chickpea genomics and breeding.
Materials and Methods
Plant Materials
The experimental material comprised 48 chickpea accessions (38 C. arietinum, 6
C. reticulatum, and 4 C. echinospermum) representing members of the primary gene
pool. The cultivated set included 26 desi and 12 kabuli accessions (Table 1). The
material was obtained from Pulse Research Laboratory, Indian Agricultural
Research Institute, New Delhi, India.
DNA Isolation and Genotyping
Genomic DNA was isolated from fresh chickpea leaves using the CTAB procedure
(Saghai-Maroof et al. 1984), quantified using a spectrophotometer, and maintained
at -20°C. Of the 100 ISSR markers tested (UBC set 9, University of British
Columbia, Vancouver, Canada), 31 polymorphic markers were analyzed in this
study. PCR amplification was conducted in 15 ll reaction volume containing 30 ng
DNA, 19 PCR buffer (10 mM Tris–HCl, pH 8.8, 50 mM KCl, and 0.1% Triton
123
123
Released variety
Genetic Stock
Genetic stock
Breeding line
Released variety
Released variety
ILC202
ILC3279
Flip87-8C
IC118913
IC296131
Advanced cultivar
ICC12968
ICCV93954
Landrace
ICC8933
Released variety
Advanced cultivar
ICC5003
ICCV96030
Landrace
ICC8159
Released variety
Landrace
ICC8151
ICCV96029
C. arietinum
Advanced cultivar
ICC4993
Released variety
Advanced cultivar
ICC4958
Released variety
Landrace
ICC4951
ICCV10
Landrace
ICC4918
ICCV88506
C. arietinum
Traditional cultivar
ICC1932
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
Traditional cultivar
ICC162
Cicer species
Biological status
Accession no.
Table 1 Source of 48 chickpea accessions used in this study
(BG203 9 P179) 9 BG303
P827 9 P9847
Breeding line
NEC141
Vyr32
[(PhuleG5 9 Narsinghpur Bold) 9 ICCC37] 9
(ICC86023-BF-BP-91-BP)
P458[(ICC5003 9 GW-GW5/7) 9 (L550 9
Gaumuchil916) 9 (ICC 1069 9 TCPS50467)]
P458[(ICC5003 9 GW-GW5/7) 9 (L550 9
Gaumuchil916) 9 (ICC 1069 9 TCPS50467)]
[(K1189 9 Chaffa) 9 G130] 9 H75
P1231 9 P1265
P458 9 [(ICC5003 9 GW-5/7) 9 (L550 9
Gaumuchil916)]
Direct selection from a genetic stock of Kanpur
Banda local 9 Etah bold
NEC2306
NEC2298
RABAT
JGC-1 (accession from M.P.)
Local selection from Nimar tract (M.P.)
Local selection from a landrace in Gulbarga (Karnataka)
P1559
P136-1
Parentage
India
India
Syria
USSR
USSR
India
India
India
India
India
India
India
India
India
USA
North Africa
India
India
India
India
India
Origin
Desi
Desi
Kabuli
Kabuli
Kabuli
Desi
Desi
Desi
Desi
Desi
Kabuli
Desi
Desi
Desi
Kabuli
Desi
Desi
Desi
Desi
Desi
Desi
Seed type
Biochem Genet
Breeding line
Genetic stock
Wild
Wild
Wild
Wild
Wild
Wild
Brachid mutant
EC556270
ILWC104
ICC17121
ICC17123
ICC17124
ICC17160
Breeding line
PG95333
Breeding line
Breeding line
BG315
IPC92-1
Genetic stock
EC539009
SBD377
C. arietinum
Released variety
IC449069
Breeding line
Released variety
IC411514
Breeding line
Released variety
IC411513
BG374
Released variety
IC296376
BG1004
C. arietinum
Released variety
IC244243
C. arietinum
C. reticulatum
C. reticulatum
C. reticulatum
C. reticulatum
C. reticulatum
C. reticulatum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
C. arietinum
Released variety
Traditional cultivar
IC244250
C. arietinum
C. arietinum
Cicer species
IC244160
Released variety
Released variety
IC296132
IC296133
Biological status
Accession no.
Table 1 continued
C. reticulatum
C. reticulatum
C. reticulatum
C. reticulatum
C. reticulatum
C. reticulatum
E100/YM
Breeding line
Breeding line
F1 (ICCV88109 9 PRR1) 9 (ICC4958)
P1013 9 BGM426 9 H-83-23
BG274 9 P436-2
BG206 9 No.501
Germplasm
[F1 (BG315 9 ILC72) 9 F1 (ICCV13 9 Flip85-11)] 9
F1 (ICCV32 9 SURUTOTO 77)]
(C104 9 BG1003) 9 (ICC88503 9 BG1048)
(IC296133 9 C. reticulatum) 9 IC296131
F1 [(IC296133 9 ICCV32)] 9 ICCV32
ICCV3 9 Flip88-120
IC296133 9 GG588
(IC296133 9 E100/YM) 9 IC296133
(ICC4951 9 850-3/27) 9 (L550 9 H208)
P1231 9 P1265
Parentage
Turkey
Turkey
Turkey
Turkey
Turkey
Syria
India
India
India
India
India
India
India
Spain
India
India
India
India
India
India
India
India
India
Origin
–
–
–
–
–
–
Desi
Kabuli
Desi
Desi
Desi
Desi
Kabuli
Kabuli
Kabuli
Kabuli
Desi
Kabuli
Kabuli
Desi
Desi
Desi
Desi
Seed type
Biochem Genet
123
Biological status
Wild
Wild
Wild
Wild
Accession no.
ILWC35
ILWC181
ILWC179
ILWC180
Table 1 continued
123
C. echinospermum
C. echinospermum
C. echinospermum
C. echinospermum
Cicer species
C. echinospermum
C. echinospermum
C. echinospermum
C. echinospermum
Parentage
Turkey
Turkey
Turkey
Turkey
Origin
–
–
–
–
Seed type
Biochem Genet
Biochem Genet
X-100), 2 mM MgCl2, 0.2 mM each dNTP, 0.5 lM each primer, and 0.5 U Taq
DNA polymerase (Bangalore Genei, Bangalore, India). Amplification reactions
were performed in a thermocycler (Biometra, Gottingen, Germany) consisting of an
initial denaturation at 94°C for 5 min, followed by 35 cycles of 50 s denaturation at
94°C, 1 min annealing at 40-50°C (depending on the primer), and 2 min extension
at 72°C. A final extension at 72°C for 7 min was also included. Amplified products
were separated on 2% agarose gels (Amresco, Solon, USA) in 19 TAE buffer,
visualized by staining with ethidium bromide, and photographed under UV light.
Size of the amplified fragments was determined using a 1 kb ladder (Fermentas Life
Science, Maryland, USA).
Data Analysis
Each ISSR fragment was considered an independent locus, and only distinct,
reproducible, and well-resolved fragments representing a consensus of independent
replicates were scored manually in a binary mode (0 for absent and 1 for present) at
a particular locus across all accessions for each primer. The potential of ISSR
markers for estimating genetic variability was examined by measuring the marker
informativeness through the counting of fragments. The total number of fragments
amplified, the number of polymorphic fragments, and the number of monomorphic
fragments were counted. To analyze the suitability of ISSR markers to evaluate
genetic profiles of chickpea, the performance of the markers was measured using
polymorphic information content (PIC; Roldan-Ruiz et al. 2000), marker index
(Varshney et al. 2007), and resolving power (Prevost and Wilkinson 1999).
The binary data matrix was used to calculate Jaccard’s similarity coefficient
between pairs of accessions using the Simqual module of NTsys-PC (Numerical
Taxonomy System version 2.1, Rohlf 2000). A similarity matrix was constructed
and further subjected to hierarchical clustering by the unweighted pair group
method with arithmetic mean (UPGMA) to generate a dendrogram for determining
the genetic diversity and relationships among the accessions. To highlight the
resolving power of the ordination, we performed a two-dimensional principal
coordinate analysis (PCoA). The clustering goodness-of-fit was evaluated by
constructing the cophenetic correlation matrix and comparing it with the similarity
matrix using Mantel’s matrix correspondence test (Mantel 1967). The test was
performed using the MXComp procedure. The result of this test is a cophenetic
correlation coefficient (r), indicating how well the dendrogram represents the
similarity data. All the above computations were performed using NTsys-PC.
Results
To investigate the genetic diversity and relationships within and among 48 chickpea
accessions, we tested 100 ISSR primers, 31 of which revealed reproducible
polymorphic patterns and were used for further analysis. Two main aspects of
genetic diversity, marker informativeness (polymorphic and overall efficiency of
informative fragment detection) and marker performance (overall efficacy of a
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Biochem Genet
Table 2 Polymorphism and marker attributes of ISSR primers used in this study
Marker
Number of fragments
Total
Monomorphic
%
Polymorphism
PIC
EMR
Marker
index
Resolving
power
0.976
Polymorphic
808
8
2
6
75
0.108
6
0.648
809
3
2
1
33.33
0.102
1
0.102
0.376
811
10
5
5
50
0.101
5
0.505
1.418
817
3
2
1
33.33
0.093
1
0.093
0.334
818
4
2
2
50
0.093
2
0.186
0.416
820
3
0
3
100
0.193
3
0.579
0.668
825
3
1
2
66.67
0.101
2
0.202
0.168
826
7
3
4
57.14
0.124
4
0.496
1.000
829
4
2
2
50
0.085
2
0.170
0.374
831
3
1
2
66.67
0.064
2
0.128
0.208
834
4
3
1
25
0.062
1
0.062
0.292
836
5
2
3
60
0.152
3
0.456
0.816
842
5
3
2
40
0.072
2
0.144
0.418
844
5
3
2
40
0.074
2
0.148
0.416
850
5
0
5
100
0.287
5
1.435
1.876
855
5
2
3
60
0.081
3
0.243
0.496
856
5
3
2
40
0.150
2
0.300
1.000
857
4
2
2
50
0.090
2
0.180
0.418
858
3
1
2
66.67
0.144
2
0.288
0.542
859
5
2
3
60
0.120
3
0.360
0.918
860
4
2
2
50
0.101
2
0.202
0.458
861
7
3
4
57.14
0.157
4
0.628
1.500
864
4
1
3
75
0.255
3
0.765
1.500
866
3
0
3
100
0.295
3
0.885
1.252
868
6
3
3
50
0.151
3
0.453
0.834
873
4
2
2
50
0.093
2
0.186
0.458
879
4
2
2
50
0.082
2
0.164
0.376
880
6
3
3
50
0.156
3
0.468
1.416
885
3
1
2
66.67
0.080
2
0.160
0.256
887
3
2
1
33.33
0.062
1
0.062
0.208
889
3
2
1
33.33
0.159
1
0.159
0.792
141
62
79
0.125
2.55
0.350
0.715
Total
Average
4.55
2
2.55
56
56.1
PIC polymorphic information content, EMR effective multiplex ratio
primer set used in determining polymorphism level, genetic diversity, and
discriminatory power) were evaluated (Table 2; Fig. 1).
Marker Informativeness
Marker informativeness of the 31 ISSR primers was analyzed using several
parameters (Table 2). Of the 141 total fragments generated across all accessions, 79
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Biochem Genet
Fig. 1 ISSR profiles of 48 chickpea accessions obtained using the primer UBC 829. Lane M 1 kb ladder
(56%) were polymorphic and 62 (44%) were monomorphic. The number of
polymorphic fragments per primer ranged from one (markers 809, 817, 834, 887,
and 889) to six (808), with an average of 2.55. The percentage polymorphism
ranged from 33.3% (809, 817, 887, and 879) to 100% (820, 850, and 866), with an
average of 56.1%. The range of frequencies of polymorphic fragments for a given
primer across all accessions was 0.02-0.98, with an average of 0.52. A large
proportion (25.3%) had frequencies in the range of 0.8-0.9 (Fig. 2).
Marker Performance
Information on the genetic profile of each accession was used to assess the marker
performance by evaluating the PIC, effective multiplex ratio (EMR), marker index,
and resolving power (Table 2). The range of PIC for the 79 polymorphic fragments
was 0.039-0.496, averaging 0.224. Eight of the polymorphic fragments were highly
informative (PIC [ 0.45), eight had low levels of PIC (\ 0.05), and the remaining 63
showed moderate values (0.05–0.45) (Fig. 3). The highest PIC value (0.295) was
observed for primer 866 and the lowest (0.062) for primers 834 and 887. The average
was 0.125. When the frequency value data were correlated with PIC value data for
individual fragments, it was found that the fragments falling within the 0.4-0.6 range
of frequency were highly informative (average PIC 0.49), followed by those in classes
Fig. 2 Frequency distribution of polymorphic ISSR fragments amplified in 48 chickpea accessions
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Biochem Genet
Fig. 3 Average PIC values for polymorphic fragments generated by ISSR primers in 48 chickpea
accessions
0.6-0.7 (average PIC 0.47) and 0.3-0.4 (average PIC 0.46) (Fig. 4). The highest
EMR (6) was observed for the primer 808, and the mean EMR per primer was 2.55. To
determine the overall utility of the marker system, we calculated the marker index for
each ISSR primer; the indices ranged from 0.062 (834 and 887) to 1.435 (850),
averaging 0.350. The resolving power, a feature that indicates the discriminatory
potential of the primer, ranged from 0.168 (825) to 1.876 (850), averaging 0.715.
Cluster Analysis
The UPGMA dendrogram was constructed from a similarity matrix based on
Jaccard’s similarity coefficient values (Fig. 5). The cophenetic correlation between
Fig. 4 Relationship between average PIC and frequency of polymorphic fragments amplified by ISSR
primers in 48 chickpea accessions
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Biochem Genet
Fig. 5 UPGMA dendrogram of 48 chickpea accessions based on Jaccard’s similarity coefficient
calculated from ISSR data set
ultrametric similarities of the tree and similarity matrix was high (r = 0.97),
indicating that the cluster analysis strongly represents the similarity data. The range
of similarity coefficient values was 0.64-1.0, suggesting a moderate level of
genetic variation. The two desi chickpea accessions ICCV93954 and SBD377 were
most closely related, having the highest similarity coefficient value (1.0). A wild
accession (ICC17124) and a cultivated accession (BG374) were the most distantly
related, with the lowest similarity coefficient value (0.64). The dendrogram clearly
grouped all the accessions into three major clusters, two representing the wild Cicer
accessions and one the cultivated chickpeas. Cluster I is composed of wild
accessions, grouping all the C. echinospermum accessions (ILWC179, ILWC180,
and ILWC181) into subcluster IA, with the exception of ILWC35 which grouped
separately with the four C. reticulatum accessions (ICC17160, ICC17121,
ICC17124, and ICC17123) in subcluster IB. It is also interesting that of the six
C. reticulatum accessions examined, two (ILWC104 and EC556270) grouped
separately in cluster II, closer to the cultivated chickpeas. Cluster III includes all the
C. arietinum accessions and is further divided into two subclusters. Subcluster IIIB
comprises five desi accessions (ICC1932, IPC92-1, ICCV10, BG374, and BG1004).
Subcluster IIIA is further divided into three groups (P, Q, and R). Groups P and Q
contain desi and kabuli accessions nondistinctively in equal share: two each in
group P (desi ICC4993 and ICC8159 and kabuli ICC8151 and ILC3279) and four
each in group Q (desi ICC162, ICCV96030, ICCV96029, and ICCV88506 and
kabuli PG95333, Flip87-8C, ICC12968, and ILC202). The ILC202 branch is
separate from the rest of group Q, indicating less similarity with these accessions.
Group R reveals a subgrouping of six kabuli accessions (EC539009, BG315,
IC449069, IC244243, IC296376, and IC411514) distinct from three desi subgroups
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Biochem Genet
of three (IC296131, IC244250, and IC296133), four (ICC4951, IC296132,
IC118913, and ICC4958), and eight (ICC8933, ICC4918, ICC5003, ICCV93954,
SBD377, IC411513, B. Mutant, and IC244160) accessions, all clearly distinguished
except ICCV93954 and SBD377. In general, the dendrogram showed separation of
desi and kabuli accessions to a great extent with few exceptions.
When the dendrogram was correlated with the pedigree data, the accessions with
similar pedigree or common parentage generally clustered together. For instance,
genotypes ICCV96029 and ICCV96030, derived from the same cross, were present in
the same group (Q) of subcluster IIIA. IC296376, IC411513, IC244160, and IC244250,
having the common parent IC296133, were present in the same group (R) of subcluster
IIIA. Likewise, IC449069 and IC296376 were present in the same subgroup of group R
in subcluster IIIA, as they have the common parent ICCV32. Genotype pairs like
BG315/IC449069, IC296131/IC411513, ICC4958/SBD377, IC296133/IC244250,
IC296133/IC244160, IC296131/IC411513, ICC5003/ICC12968, and ICC4951/
IC296133 were closely related and present in the same subcluster (IIIA), as the first
genotype of each pair was one of the parents of the second. There was a lack of
correlation, however, between the grouping of accessions and their geographic origin.
Principal Coordinate Analysis
The genetic similarity matrix based on Jaccard’s similarity coefficient was also
subjected to PCoA, for better visualization of the genetic structure and relationships
among the accessions (Fig. 6). The results were in accordance with the cluster
Fig. 6 Two-dimensional plot obtained from principal coordinate analysis of 48 chickpea accessions
using ISSR markers
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Biochem Genet
analysis to a great extent. Two-dimensional dispersion showed that all the wild
Cicer accessions were clearly distinguished and nested apart from the cultivated
accessions in cluster I, except two: the C. reticulatum accessions ILWC104 and
EC556270 formed a separate cluster (cluster II). Because of their low genetic
variation, all the cultivated accessions formed a separate intensive group (cluster
III), except for five desi accessions, ICC1932, IPC92-1, ICCV10, BG374, and
BG1004, which formed a separate small subcluster of the third major cluster at the
top left, concordantly with the dendrogram.
Discussion
Understanding genetic diversity and genetic relationships in germplasm collections
is critical to crop improvement programs. Chickpea has a narrow genetic base
(Abbo et al. 2003; Nguyen et al. 2004) in spite of a large collection of germplasm
and a globally active genetic enhancement program; this probably is due to the
utilization of a few closely related varieties for hybridization. The use of diverse
materials from the primary gene pool (C. arietinum, C. echinospermum, and C.
reticulatum), including desi 9 kabuli and interspecific crosses with wild relatives,
coupled with induced mutagenesis to incorporate valuable genes (Glaszmann et al.
2010), may lead to a broadening of the genetic resource base. It is expected that the
use of such diverse lines will improve the chances of the appearance of transgressive
segregants with beneficial traits, because of the reshuffling of alleles through
recombination. High-yielding varieties with desirable trait combinations, such as
improved grain quality and enhanced resistance to various biotic and abiotic
stresses, can thus be selected from these segregants.
The 31 ISSR markers evaluated in 48 chickpea accessions revealed moderate
levels of diversity, detecting a total of 141 fragments, 79 of them polymorphic,
averaging 2.55 polymorphic fragments per primer. This level of polymorphism
(56%) is higher than the level (26%) reported by Chowdhury et al. (2002) and lower
than that (82%) of Rao et al. (2007).
Our study also evaluated the informativeness or discriminatory power of ISSR
primers for genetic diversity studies through the PIC, marker index, EMR, and
resolving power, features that to the best of our knowledge have not yet been
reported in other ISSR studies in chickpea. The ISSR primers generated ten highly
informative polymorphic loci (PIC [ 0.4) among 79 polymorphic fragments
(Fig. 3). The highest PIC (0.295) was found for primer 866, which is therefore
recommended for germplasm analysis. Similar to results in other crops (RoldanRuiz et al. 2000; Varshney et al. 2007; Grativol et al. 2011), ISSR fragments
amplified with the frequency range of 0.4–0.6 proved to be the most informative,
followed by 0.6-0.7 and 0.3-0.4. Hence, targeting fragments in these classes is
recommended for diversity analysis in case a large number of fragments are
detected by a particular primer set. The marker index varied from 0.062 to 1.435
(average 0.350) and has been used to assess the informativeness of various markers
in several crop species, including soybean (Powell et al. 1996), wheat (Bohn et al.
1999), corn salad (Muminovic et al. 2004), and jatropha (Grativol et al. 2011).
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Biochem Genet
Resolving powers in our study were in the range of 0.168-1.876 (average 0.715)
per primer. Prevost and Wilkinson (1999) and Fernandez et al. (2002) detected a
strong and linear relationship between the ability of a primer to distinguish
accessions and resolving power values, suggesting that in our study, primer 850,
with the highest resolving power (1.876), should be the most informative primer for
distinguishing the accessions.
In our study, the coefficient of similarity ranged from 0.64 to 1.0. The highest
genetic similarity was between two cultivated chickpea accessions, ICCV93954 and
SBD377, with a similarity coefficient of 1.0. The most diverse accessions, on the two
extremes of the dendrogram, were the cultivated BG374 and wild ICC17124, with a
similarity coefficient of 0.64. A similar range of genetic similarity was reported by
Rao et al. (2007) using ISSR markers in chickpea. Our UPGMA dendrogram
separated all the chickpea accessions into three major clusters, two (cluster I and II)
representing wild accessions and the third (cluster III) the cultivated chickpea, and
the PCoA displayed a similar profile of major clusters, with minor deviations. Rao
et al. (2007) reported a similar profile, with clusters clearly discriminating the wild
accessions from cultivated chickpeas. In the PCoA plot, five kabuli accessions
(ICC1932, IPC92-1, ICCV10, BG374, and BG1004) clustered together and swerved
a little from the other cultivated accessions, indicating their distinct identity, which
can be explained by the conscious selection criteria adopted in developing these
varieties for certain domesticated traits. These accessions could be of interest for
mapping purposes and can be included in crossing programs to broaden the genetic
base of chickpea. It is also interesting that in the UPGMA and PCoA, two
C. reticulatum accessions, ILWC104 and EC556270, did not group with other
C. reticulatum accessions, forming instead a separate cluster (II), indicating genetic
dissimilarity of these accessions from the other C. reticulatum accessions. Cultivated chickpeas were found to be closer to C. reticulatum than C. echinospermum, a
result corroborated by several previous studies using other molecular markers
(Sudupak et al. 2002; Nguyen et al. 2004; Sudupak 2004; Rao et al. 2007; Choudhary
et al. 2012).
Despite the presence of a high degree of relatedness among the cultivar pairs, all
were clearly distinguished except two, ICCV93954 and SBD377. ISSR analysis
divided the cultivated chickpeas into two subclusters (IIIA and IIIB) on the basis of
seed morphology; hence, our study is supported by other studies that differentiate
the cultivated chickpea into two gene pools, desi and kabuli (Upadhyaya et al. 2008;
Sefera et al. 2011; Choudhary et al. 2012). Additionally, the genetic relationships
correlated with the known pedigree information, grouping the accessions derived
from the same cross or having a common parent in the same subclusters. The
chickpea accessions, however, did not strictly group according to geographic origin.
These results are indicative of extensive germplasm exchange among geographic
regions.
The results of the present ISSR analysis were similar but not identical to our
earlier SSR study of the same set of accessions (Choudhary et al. 2012). The
differences may be attributed to the different numbers of loci analyzed and to
differences in the nature of the marker systems analyzed, reinforcing the importance
of the number and nature of the loci examined and their overall coverage of the
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genome in obtaining reliable estimates of the genetic diversity and relationships
among the accessions. The SSR markers identified in our earlier study and the ISSR
markers from this study should complement one another during genetic identification, in that they cover different regions of the chickpea genome. Consistent with
the earlier study, this study revealed low genetic diversity in C. arietinum compared
with its wild relatives, supporting the conclusion that chickpea has a narrow genetic
base (Abbo et al. 2003; Nguyen et al. 2004). Hence, it is vital to broaden the genetic
base of cultivated chickpea by using wild species of the primary gene pool, which
hold a wealth of new alleles. If included in breeding programs, they can help raise
yield levels, quality, and stress resistance in the cultivated chickpea (Berger et al.
2003; Nguyen et al. 2004; Singh et al. 2008). Recent studies have shown a huge
amount of genetic diversity in the primary gene pool (Berger et al. 2003; Singh et al.
2008), which is being utilized through interspecific hybridization, but a significant
amount has still not been exploited, and it can be used to enhance diversity and
performance under diverse agro-ecological conditions.
In conclusion, this study indicates that ISSR proved to be an efficient marker
system for studying genetic diversity and relationships among members of the
primary gene pool. It will serve as an important consideration for efficient
rationalization and utilization of the primary gene pool, providing a basis for future
chickpea crop variety identification, conservation, and management. The promising
accessions identified through this study will serve as useful resources for functional
and comparative genomics, in mapping and cloning genes, and in applied breeding
for enhancing the genetic potential of the chickpea.
Acknowledgments The authors gratefully acknowledge the Indian Council of Agricultural Research
(ICAR) Network Project on Transgenics in Crops (Functional Genomics component) for providing the
financial support for this study.
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